Open AccessAddressing Reported Pro-Apoptotic Functions of NF-κB: Targeted Inhibition of Canonical NF-κB Enhances the Apoptotic Effects of Doxorubicin
Author(s) -
Brian Keith Bednarski,
Albert S. Baldwin,
Hee Jin Kim
Publication year - 2009
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0006992
Subject(s) - doxorubicin , apoptosis , nf κb , cancer research , biology , microbiology and biotechnology , downregulation and upregulation , iκbα , viability assay , chemistry , biochemistry , chemotherapy , gene , genetics
The ability of the transcription factor NF-κB to upregulate anti-apoptotic proteins has been linked to the chemoresistance of solid tumors to standard chemotherapy. In contrast, recent studies have proposed that, in response to doxorubicin, NF-κB can be pro-apoptotic through repression of anti-apoptotic target genes. However, there is little evidence analyzing the outcome of NF-κB inhibition on the cytotoxicity of doxorubicin in studies describing pro-apoptotic NF-κB activity. In this study, we further characterize the activation of NF-κB in response to doxorubicin and evaluate its role in chemotherapy-induced cell death in sarcoma cells where NF-κB is reported to be pro-apoptotic. Doxorubicin treatment in U2OS cells induced canonical NF-κB activity as evidenced by increased nuclear accumulation of phosphorylated p65 at serine 536 and increased DNA–binding activity. Co-treatment with a small molecule IKKβ inhibitor, Compound A, abrogated this response. RT–PCR evaluation of anti-apoptotic gene expression revealed that doxorubicin-induced transcription of cIAP2 was inhibited by Compound A, while doxorubicin-induced repression of other anti-apoptotic genes was unaffected by Compound A or siRNA to p65. Furthermore, the combination of doxorubicin and canonical NF-κB inhibition with Compound A or siRNA to p65 resulted in decreased cell viability measured by trypan blue staining and MTS assay and increased apoptosis measured by cleaved poly (ADP-ribose) polymerase and cleaved caspase 3 when compared to doxorubicin alone. Our results demonstrate that doxorubicin-induced canonical NF-κB activity associated with phosphorylated p65 is anti-apoptotic in its function and that doxorubicin-induced repression of anti-apoptotic genes occurs independent of p65. Therefore, combination therapies incorporating NF-κB inhibitors together with standard chemotherapies remains a viable method to improve the clinical outcomes in patients with advanced stage malignancies.